PROJECT SUMMARY/ABSTRACT Arsenic contamination of groundwater remains a widespread public health crisis, affecting an estimated 150 million people worldwide, including 13 million in the United States. Even at low doses, chronic arsenic exposure leads to multiple morbidities and mortalities. A growing body of literature demonstrates the health impacts of in utero arsenic exposure resulting in long term increases in morbidity, a prime example of which is increased risk for chronic respiratory disease (CRD). Whereas many of the mechanisms are unclear, it is known that in utero arsenic exposure leads to pulmonary hypoplasia, or decreased lung growth. Using a microfluidic ex vivo culture model of the embryonic lung, we can culture lung explants in a physiologically relevant mechanical environment and simultaneously interrogate molecular and mechanical events with high temporal and spatial resolution. Our findings have demonstrated that branching is regulated by coordinated airway smooth muscle (ASM) contractions and dysregulation has major functional consequences on lung development. Additionally, we determined that arsenic alters expression of core Hippo pathway components that are known to regulate organ size. We hypothesize that fetal arsenic exposure impairs airway morphogenesis through two independent mechanisms: dysregulation of active smooth muscle contractions that guide development and hyperactivation of the Hippo growth control pathway within the airway epithelium to cause hypoplasia. We will investigate this hypothesis using our novel microfluidic embryonic organ explant culture model in two independent aims. In our first Aim, we test the effect of arsenic on the coordinated ASM contractions that are tightly coupled to airway branching and lung growth. Whereas the function of ASM contractions are incompletely understood, it is known that ASM contractions require Ca2+ signaling. Ca2+ signaling is disrupted by arsenic in other smooth muscle tissues. Our preliminary data support dysregulated Ca2+ signaling in the ASM to cause a hypercontracted phenotype and hypoplasia. In our second Aim, we test the importance of Hippo signaling as a mediator of lung growth, using a combination of molecular signaling perturbation and morphometric techniques. Hippo signaling has been implicated in controlling organ size in a variety of systems, and improper activity can lead to dramatic hypoplasia. However, Hippo signaling has been minimally investigated in lung development. Additionally, arsenic induced activation of Hippo has been shown in mouse epidermis. In both Aims, we will target our hypothesized mediators of arsenic toxicity using either an FDA approved compound (isoprotenerol) or the recently developed small-molecular inhibitor (XMU-MP-1), potentially offering a path to prophylactic treatments for at-risk populations. In aggregate, these studies will increase understanding of the effects of arsenic exposure on early lung development, inform new policies and EPA exposure limits for expectant mothers, and hold promise to rapidly translate these findings to improve prenatal health.